Growth factors and their receptors are involved in the regulation of cell proliferation, and they also seem to play a role in tumor growth. The c-erbB-2 growth factor receptor protein, a protein of the membrane receptor protein tyrosine kinase family (A. Ullrich & J. Schlessinger, Cell 61: 203-212, 1990), is found in human breast tumors and human ovarian carcinomas. Amplification of the c-erbB-2 gene and over-expression of the protein appears to correlate with poor prognosis for tumor patients. Thus the c-erbB-2 protein has potential, both as a diagnostic marker and as a target for cancer therapy. Sequence analysis reveals that c-erbB-2, also called HER2, a glycoprotein of 185 kilo-Dalton (gp185), is identical or closely related to the human analog of the neu oncogene (A. L. Schechter et al., Science 229: 976-978, 1985) and shows considerable sequence homology to the human epidermal growth factor (EGF) receptor.
Of particular interest in tumor diagnosis and therapy are antibodies directed to tumor markers. Polyclonal antibodies may be obtained from the serum of mammals immunized with the antigen, i.e. the tumor marker. The development of hybridoma technology made it possible to generate continuous cell lines, in particular murine hybridomas, producing monoclonal antibodies of the desired specificity. Murine monoclonal antibodies directed to c-erbB-2 are known and are described, for example, by S. J. McKenzie at al., Oncogene 4: 543-548, 1989; R. M. Hudziak et al., Molecular and Cellular Biology 9: 1165-1172, 1989; International Patent Application WO 89/06692 (Genentech); and Japanese Patent Application Kokai 02-150 293 (Ajinomoto KK).
A major limitation in the use of murine-derived monoclonal antibodies as in vivo diagnostic and therapeutic agents is their immunogenicity as foreign proteins, their rather long persistence in the circulation, and the formation of damaging immune complexes. On the other hand, the treatment with human monoclonal antibodies is also limited since human hybridoma cell lines are hard to prepare, generally unstable, and do not produce monoclonal antibodies of appropriate specificity in sufficient quantities and at reasonable costs. In principle, the in vitro use of murine monoclonal antibodies is without limitation. However, production costs of monoclonal antibodies and, depending on the type of immunoassay used, the need for attaching a detectable marker to the antibody make it desirable to find more economic alternatives to regular murine monoclonal antibodies.
A promising alternative is the modification of immunoglobulin genes in order to tailor antibodies for particular diagnostic and therapeutic tasks. Due to the fact that the variable region and each of the constant region domains of immunoglobulin molecules are encoded in separate exons with their own splice sites, recombinant DNA techniques can be used to isolate different parts of cloned immunoglobulin genes and ligate them to parts of other immunoglobulins or to effector molecules. The reconstructed genes are expressed by appropriate transformed continuous cell lines. Murine antibodies can, for example, be converted into "humanized" antibodies by exchanging murine constant domain exons for human immunoglobulin constant domain exons, thus generating chimeric antibodies with murine antibody-combining sites and human constant domains. The chimeric antibodies retain the antigen specificity determined by the murine variable domains, but also exhibit human effector functions (such as complement binding, stimulation of phagocytosis, triggering of granule release by mast cells) determined by the carboxy-terminal constant domain segments of the heavy chain polypeptides. An even more sophisticated technique in tailoring antibodies described in European Patent Application 0 239 400 exchanges also other fairly conserved domains, the so-called framework regions (FRs), within the murine variable domains for corresponding framework regions from human antibodies or for other human protein sequences. Such an antibody should be even less immunogenic in man since the only parts derived from a murine antibody are those hypervariable regions which define a particular specificity for an antigen, the so-called complementarity determining regions (CDRs).
Furthermore, fusion proteins different from immunoglobulins may be formed, e.g. single-chain antibodies, which retain the specificity and binding properties of the starting murine monoclonal antibody, but have otherwise novel properties derived from the non-immunoglobulin part of the fusion protein. The smallest domain of a monoclonal antibody which can bind to the antigen is the so-called Fv fragment which consists of the variable domains of the heavy and light chains. Fv fragments are difficult to prepare by proteolytic techniques since the corresponding variable domains tend to dissociate upon dilution. Fv molecules constructed by joining the variable domains of the heavy and light chains via a short peptide linker, also called single-chain antigen binding proteins, bind to an antigen with similar characteristics as the original monoclonal antibody (R. E. Bird et al., Science 242: 423-426, 1988; J. S. Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883, 1988; and International Patent Application WO 89/09825 (Celltech)). Fv encoding genes can, in principle, be linked to genes encoding effector molecules by recombinant gene technology. It is known, for example, that Fv encoding gene sequences can be linked to a gene encoding a portion of the Pseudomonas exotoxin A gene (V. K. Chaudhary et al., Nature 339: 394-397, 1989; and International Patent Application WO 89/11533 (I. Pastan et al.)).